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Oxidative Stress and Antioxidant Defense Mechanism in Glomerular Diseases
Free Radical Biology & Medicine, Vol. 22, Nos. 1/2, pp. 161–168, 1997 Copyright q 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(96)00284-5
Original Contribution OXIDATIVE STRESS AND ANTIOXIDANT DEFENSE MECHANISM IN GLOMERULAR DISEASES Sa´ndor Tu´ri,* Ilona Ne´meth,* Attila Torkos,* Levente Sa´ghy,* Ilona Varga,† Be´la Matkovics,† and Judit Nagy‡ *Department of Paediatrics, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; †Department of Biology, Attila Jo´zsef University, Szeged, Hungary; and ‡2nd Department of Medicine, Medical University, Pe´cs, Hungary (Received 21 November 1995; Revised 26 April 1996; Accepted 9 May 1996)
Abstract—The changes in red blood cell (RBC) lipid peroxidation [measured via the malonyl dialdehyde (MDA) concentration], reduced (GSH), and oxidized glutathione (GSSG) levels, hemoglobin (Hb) oxidation and antioxidant enzyme [catalase (Cat), glutathione peroxidase, and superoxide dismutase (SOD)] activities were studied in 45 pediatric patients with various glomerular diseases [minimal change nephrotic syndrome (MCNS) in relapse or in remission, lupus nephropathy (SLE), poststreptococcal glomerulonephritis (APSGN), IgA nephropathy (IGA gn)], and in 20 adult patients with IGA gn and also in 15 pediatric and 14 adult controls. The in vitro effects of hydrogen peroxide [acetyl phenylhydrazine (APH) test] on the GSH and Hb metabolisms were likewise investigated. There was an increased oxidative stress in MCNS with relapse, IGA gn, SLE gn, and APSGN, which could be detected in the GSH and Hb oxidation and in the lipid peroxidation on the peripheral RBC-s. The RBC SOD and Cat activities were significantly lower in all patients than in the controls. The RBC GSSG level was significantly elevated in all patients, with the exception of MCNS in remission. This stimulated a compensatory GSH production in MCNS with relapse and in IGA gn, but not in SLE or APSGN. The regeneration of GSH from GSSG was reduced in MCNS with relapse, SLE, and IGA gn, but not in APSGN. In remission, the GSH-GSSG redox system normalizes, but in vitro the APH test stimulates an intensive Hb oxidation. In conclusion, there is a correlation between the presence of active glomerular disease and the evidence of oxidative changes in the various parameters measured in peripheral RBCs. Copyright q 1996 Elsevier Science Inc. Keywords—Antioxidant defense, Glomerular disease, Oxidative stress, Free radicals
puromycin aminonucleoside (PAN)-induced nephrosis,5,6 murine lupus nephritis,7 and adriamycin-induced nephrosis.8 However, the role of oxidative injury in glomerular diseases was not confirmed by other groups.9,10 The study by Alfrey et al. indicated that the increased urine iron excretion is associated with hydroxyl radical production, which causes tubular injury.11 Vitamin E treatment was earlier found to cause an increased renal protein SH production, a decreased protein excretion, and an improved creatinine clearance in the heterologous phase of antiGbm nephritis.12 A selenium-deficient diet resulted in a reduced glutathione peroxidase activity (GP-ase) and an increased urine protein excretion.13 Fish oil feeding of rats with murine lupus nephritis resulted in an undetectable level of proinflammatory cytokines and an increased antioxidant enzyme gene ex-
INTRODUCTION
A still growing body of evidence has accumulated, indicating that a disturbance of the balance between oxidative stress and antioxidant defense mechanisms plays a major role in the pathomechanism of glomerular diseases. In antiglomerular basement membrane nephritis (anti-Gbm nephritis), the plasma lipid peroxidation is increased,1 and the administration of antioxidant enzymes [superoxide dismutase (SOD) and catalase (Cat)] resulted in a reduction of renal malonyl dialdehyde (MDA) production and urine protein excretion; further morphological protection was detected.2–4 Similar findings were observed in Address correspondence to: Sa´ndor Tu´ri M.D., Department of Paediatrics Albert Szent-Gyo¨rgyi Medical University, Kora´nyi Street 14, 6725 Szeged, Hungary. 161
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S. TURI et al. Table 1. MDA Concentrations in the Plasma and RBC (mean { SD)
PATIENTS AND METHODS
nM MDA/mg protein Dg
n
Cont.ped. Cont.adult APSGN SLE MCNS rel. MCNS rem. IGA ped. IGA adult
15 14 8 7 15 17 4 20
plasma 0.189 0.202 0.257 0.244 0.332 0.196 0.240 0.244
{ { { { { { { {
RBC
0.017 0.019 0.055* 0.044* 0.030† 0.053 0.046* 0.041*
0.350 0.356 0.486 0.379 0.454 0.439 0.430 0.438
{ { { { { { { {
0.030 0.025 0.010† 0.016* 0.010† 0.012† 0.060* 0.070*
APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * p õ 0.05. † p õ 0.01.
pression.14 The results of Andreoli et al.15 and Yagucchi et al.16 suggested that reactive oxygen species could be induced by glomerular resident cells. It appeared that infiltration of neutrophils in the glomeruli might not be related to proteinuria and glomerular injuries in the first phase of Masugi nephritis. It was postulated that massive proteinuria in the first phase of Masugi nephritis might be correlated with the activities of reactive oxygen species induced by the glomerular cells.16 Apart from these experimental results, only scanty clinical data are available on the roles of oxidative stress and antioxidant defense mechanisms in the pathogenesis of glomerular diseases. The aim of the present study was to investigate the changes in lipid peroxidation, the red blood cell (RBC) glutathione redox system, hemoglobin (Hb) oxidation and antioxidant enzyme activities in glomerular diseases of pediatric and adult patients.
Forty-five pediatric (25 boys and 20 girls) and 20 adult patients (12 males and 8 females) were investigated in comparison with 15 pediatric and 14 adult age and sex-matched controls. The median ages of the patients were as follows: children 8 years (range 3–15 years), adults 31 years (21–45 years). The distribution of the clinical subgroups among the pediatric patients: acute poststreptococcal glomerulonephritis (APSGN): 8; systemic lupus erythematodes associated with nephrotic syndrome (SLE): 7; minimal change nephrotic syndrome in relapse (MCNS rel): 15; minimal change nephrotic syndrome in a remission of õ2 months’ duration (MCNS rem): 17; IgA nephropathy (IGA ped.): 4 patients. All of the adult patients had IgA nephropathy (IGA adult). The nephrotic patients (MCNS rel., IGA ped. or SLE) displayed ú 2 g protein excretion daily. Individuals belonging in the APSGN and IGA adult subgroups had ú 200 RBC-s/ml urine. Seven adult IGA patients exhibited ú 2 g/d urine protein excretion. Ten members of the adult IGA subgroup required antihypertensive therapy (calcium channel blockers and/or converting enzyme inhibitors). The pediatric patients received no antihypertensive treatment. The nephrotic children (MCNS rel. and IGA) participated in Prednisolone therapy (60 mg/m2 body surface area daily in the first month, and 35 mg/m2 every second day in the second month). The SLE patients received chlorambucil (0.2 mg/kg body weight daily), or azathioprine (2 mg/kg/d) together with low-dose Prednisolone. None of the patients were given nonsteroidal antiinflammatory drugs, albumin or blood transfusions in the 2-week period prior to the investigation. The study was approved by the University Research Ethical Committee. After informed consent had been obtained, blood
Table 2. RBC Antioxidant Enzyme Activities (mean { SD) U/mg protein 1 1002 Dg
n
Cont.ped Cont.adult APSGN SLE MCNS rel. MCNS rem. IGA ped. IGA adult
15 14 8 7 15 17 4 20
GP-ase 0.91 1.02 1.06 0.81 0.89 0.91 0.90 0.89
{ { { { { { { {
0.06 0.06 0.3 0.07 0.06 0.21 0.10 0.14
SOD 1.32 1.30 0.256 0.220 0.553 0.213 0.337 0.348
{ { { { { { { {
0.12 0.32 0.018* 0.08* 0.39* 0.13* 0.03* 0.02*
Cat 9.48 10.2 5.14 5.67 6.60 5.52 5.64 5.61
{ { { { { { { {
1.9 1.4 1.9* 1.8* 0.9* 2.03* 1.1* 1.7*
APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * p õ 0.01.
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Fig. 1. Red blood cell oxidized glutathione (GSSG) concentrations in different glomerular diseases (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IgA nephropathy; ped.: pediatric. ** Å p õ 0.01.
samples were drawn into tubes with EDTA and heparin for the following studies.
with 5 mg/ml acetylphenylhydrazine (APH) at 377C for 1 h. The residual GSH was expressed as a percentage of the original concentration.
GSH determinations The concentrations of reduced (GSH) and oxidized glutathione (GSSG) in the whole blood hemolysate were measured by means of the highly sensitive method of Tietze.17 This assay responds to both GSH and GSSG, so GSSG was determined separately after the alkylation of GSH with N-ethylmaleimide (NEM) according to the method of Akerboom and Sies.18 GSSG was separated from NEM by gel filtration with Sephadex G-10 (Pharmacia, Uppsala, Sweden).19 Both GSH and GSSG were related to the Hb contents of the haemolysates.
Antioxidant enzyme and MDA studies Heparinized blood was used for MDA and antioxidant enzyme activity measurements in parallel with GSH and GSSG determinations. The methods for the measurement of SOD,21,22 Cat,23 GP-ase23,24 and MDA25 activities were described in detail previously.26 The protein concentration of RBC-s was determined with the Folin reagent.27 The appearance of Hb derivative
GSH instability test This was carried out by the method of Beutler.20 After GSH determination, whole blood was incubated
In in vitro tests, the changes in the level of hemichrome during oxidative stress were examined by the method of Szebeni et al.28 before and after APH incu-
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Fig. 2. Red blood cell reduced glutathione (GSH) levels in different glomerulopathies (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * Å p õ 0.05.
bation. All determinations were carried out in duplicate (two samples from each patient). Statistical analyses were performed with the paired t-test and Spearman rank correlation test. The results for the groups are given as mean values plus or minus SD.
A positive correlation was found between the RBC and plasma MDA concentrations (r Å 0.59, p õ 0.05) (Table 1). There were no significant differences in RBC GPase activities between patients and controls. However, the SOD and Cat values were significantly lower in all patients than in the controls (p õ 0.01) (Table 2).
RESULTS
MDA and antioxidant enzyme studies
RBC GSH-GSSG determinations
The RBC MDA results were elevated in all patient groups as compared with the controls (SLE, IGA ped., IGA adult p õ 0.05; MCNS rel., MCNS rem., APSGN p õ 0.01). Similarly, the plasma MDA values were also significantly higher in all patient groups (with the exception of MCNS rem) than in the controls (MCNS rel p õ 0.01, APSGN, SLE, IGA ped., IGA adult p õ 0.05).
As compared with the controls, the GSH oxidation (GSSG concentration) was significantly higher in all patient groups (p õ 0.01) with the exception of MCNS rem (Fig. 1). We observed a compensatory increase of GSH in the MCNS rel and IGA ped, groups as compared with the pediatric controls (p õ 0.05). Nevertheless, in APSGN this level was significantly lower (p õ 0.05).
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Fig. 3. Red blood cell glutathione redox ratio (GSSG/GSH %) in glomerular diseases (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. ** Å p õ 0.01.
The other groups did not differ from their age-matched controls (Fig. 2). The GSSG/GSH ratio was significantly higher in APSGN, SLE, and MCNS rel. as compared with the other patient groups and the controls (p õ 0.01) (Fig. 3). In the APH test the GSH regeneration was significantly lower in the groups of SLE (p õ 0.001), MCNS rel. and IGA adult (p õ 0.05) than in the controls. In response to hydrogen hyperoxide, a significantly higher productions of hemichrome was observed in all patients’ groups (SLE p õ 0.001, APSGN, MCNS rel. p õ 0.01, MCNS rem., IGA adult and ped. p õ 0.05) than in the age-matched controls. It is noteworthy that the adult controls gave significantly higher values than the pediatric controls (p õ 0.05) (Fig. 4). DISCUSSION
Reactive oxygen molecules participate in the pathogenesis of various renal diseases, including inflamma-
tory lesions such as glomerulonephritis and interstitial nephritis, ischemic reperfusion injury, hemolytic uremic syndrome and toxic nephropathies, and possibly in the progression of chronic renal failure. These reactive oxygen species, including superoxide anion, hydrogen peroxide, hydroxyl radical, hypochlorous acid, and peroxynitrite, may be generated by activated neutrophils, monocytes, and mesangial cells during metabolic processes.29 Oxidant stress may induce injury to proteins by oxidation of critical amino acids, resulting in a loss of enzymatic activity or structural integrity. Oxidant-induced lipid peroxidation causes a loss of membrane stability and integrity, leading to an increased transepithelial permeability. The mechanisms of cell injury that lead to cell death following oxidant stress in cells are complex and multifactorial and may differ according to cell type. We have demonstrated an increased oxidative stress in IGA gn, SLE, APSGN, and in MCNS with relapse, which was detected in the GSH and Hb oxidation on peripheral RBC-s and lipid
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Fig. 4. The changes of reduced glutathione stability and hemoglobin oxidation after an in vitro oxidation stress. APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * Å p õ 0.05, ** Å p õ 0.01, *** Å p õ 0.001, § Å p õ 0.05 in controls.
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Oxidant injury in glomerulopathies
peroxidation in RBC-s and plasma. There was no difference in GSH and Hb oxidation and lipid peroxidation among these various forms of active glomerular diseases. The superoxide radical plays a major role in the neutrophil-mediated acute inflammatory response. Neutrophils produce superoxide for the primary purpose of aiding in the killing of microbes. The superoxide released from actively phagocytosing neutrophils serves to attract more neutrophils by reacting with and activating a latent chemotactic factor present in the plasma30 and results in extensive tissue damage. By preventing the activation of this superoxide-dependent chemotactic factor, SOD exerts potent antiinflammatory action. It is very likely that our MCNS patients produce the superoxide radical via their glomerular cells. In our study, the RBC SOD and Cat activities were significantly lower in all patients, including MCNS in rem, than in the controls. The increased GSH oxidation stimulates a compensatory GSH production in MCNS rel and IGA gn, but not in SLE or APSGN. The GSSG/ GSH ratio was the highest in SLE, APSGN and MCNS rel, which reflects an increased oxidative stress. The blood glutathione redox ratio is a parameter of the oxidative stress.31 The regeneration of GSH from GSSG was reduced in MCNS rel, SLE, and adult IGA gn. Simultaneously, an increase in the hemichrome percentage was observed in the same groups of patients. The insufficient regeneration of GSH resulted in an increased production of irreversibly oxidized Hb. The MCNS patients in remission featured a markedly decreased GSSG production and GSSG/GSH redox ratio. In the APH test the GSH stability improved and the percentage of hemichrome decreased resulting in a better Hb protection. This relates to a more specific marker role of the GSH system, in measuring the activity of oxidative stress. As far as the role of Prednisolone, or the other immunosuppressive drugs (chlorambucil and azathioprine) in the increased oxidative stress is concerned, further studies are required. At least no correlation was observed between the dose of these drugs and the level of GSH and Hb oxidation or MDA concentration. APSGN patients had similar results as the nephrotic patients treated with immunosuppressive therapy. Lastly, MCNS patients in remission and without Prednisolone had similarly elevated level of RBC MDA and low activity of RBC SOD and Cat as it was observed in MCNS with relapse. These clinical data yield multiple evidence that oxidative stress is present in the development of glomerular disease, although there are differences between the histological groups in the compensatory reactions of
167
the GSH-GSSG redox system. In MCNS the source of the free oxygen radicals could be glomerular cells, while in the other forms the role of activated neutrophils or mononuclear cells cannot be excluded. In conclusion, antioxidant therapy and diet might be beneficial in the management of these glomerular diseases. Acknowledgements — This study was supported by Hungarian ETT (525/9306) and OTKA (T01 7476) research grants.
REFERENCES 1. Hattori, T.; Ito, M.; Suzuki, Y. Changes in intra-renal scavenging activities of the reactive oxygen species in experimental glomerulonephritis and nephrosis in rats. Nippon Jinzo Gakkai Shi 33:191–199; 1991. 2. Rehan, A.; Johnson, K. J.; Wiggins, R. C.; Kunkel, R. G.; Ward, P. A. Evidence for the role of oxygen radicals in acute nephrotoxic nephritis. Lab. Invest. 51:396–403; 1984. 3. Adachi, T.; Fukuta, M.; Ito, Y.; Hirano, K.; Sugiura, M.; Sugiura, K. Effect of superoxide dismutase on glomerular nephritis. Biochem. Pharmacol. 35:341–345; 1986. 4. Birtwistle, R. J.; Michael, J.; Howie, A. J.; Adu, D. Reactive oxygen products in heterologous anti-glomerular basement membrane nephritis in rats. Br. J. Exp. Pathol. 70:207–213; 1989. 5. Diamond, J. R.; Bonventre, J. V.; Karnovsky, M. J. A role for oxygen free radicals in aminonucleoside nephrosis. Kidney Int. 29:478–483; 1986. 6. Higuchi, A. Effects of human Cu, Zn-superoxide dismutase in aminonucleoside nephrosis—Evaluation of the morphology and glomerular basement membrane anionic charge sites. Nippon Jinzo Gaggai Shi 32:767–775; 1990. 7. Sato, T. Role of active oxygen on the progression of murine lupus nephritis. Nippon Jinzo Gaggai Shi 33:239–246; 1991. 8. Wu, S. H.; Yang, Y. C.; Wang, Z. M. Role of oxygen radicals in adriamycin-induced nephrosis. Chin. Med. J. Eng. 103:283– 289; 1990. 9. Webb, D. B.; Mackenzie, R.; Zoob, S. N.; Rees, A. J. Evidence against a role for superoxide ions in the injury of nephrotoxic nephritis in rats. Clin. Sci. 69:687–689; 1985. 10. Bertolatus, J. A.; Klinzman, D.; Bronseman, D. A.; Ridnour, L.; Oberley, L. W. Evaluation of the role of reactive oxygen species in doxorubicin hydrochloride nephrosis. J. Lab. Clin. Med. 118:435–445; 1991. 11. Alfrey, A. C.; Froment, D. H.; Hammond, W. S. Role of iron in the tubulointerstitial injury in nephrotoxic serum nephritis. Kidney Int. 36:753–759; 1989. 12. Endreffy, E.; Tu´ri, S.; La´szik, Z.; Bereczki, Cs.; Ka´sa, K. The effects of vitamin E on tissue oxidation in nephrotoxic (antiglomerular basement membrane) nephritis. Pediatr. Nephrol. 5:312–317; 1991. 13. Baliga, R.; Baliga, M.; Shah, S. V. Effect of selenium-deficient diet in experimental glomerular disease. Am. J. Physiol. 263:F56–61; 1992. 14. Chandrasekar, B.; Fernandes, G. Decreased pro-inflammatory cytokines and increased antioxidant enzyme gene expression by omega-3 lipids in murine lupus nephritis. Biochem. Biophys. Res. Commun. 200:893–898; 1994. 15. Andreoli, S. P. Reactive oxygen molecules, oxidant injury and renal disease. Pediatr. Nephrol. 5:733–742; 1991. 16. Yaguchi, Y.; Tomino, Y.; Ozaki, T.; Okumura, K.; Sendo, F.; Koide, H. Correlation between reduction of polymorphonuclear leucocytes in glomeruli injected with a newly developed monoclonal antineutrophil antibody and proteinuria in Masugi nephritis. Nephron 62:444–448; 1992. 17. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Application to mammalian blood and other tissues. Anal. Biochem. 27:502–522; 1969.
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18. Akerboom, T. P. M.; Sies, H. Assay of glutathione, glutathione disulphide and glutathione mixed disulphides in biological samples. Methods Enzymol. 77:373–382; 1981. 19. Guntherberg, H.; Rapaport, S. Eine Methode zur Bestimmung des oxidierten Glutathions. Acta. Biol. Med. Germ. 20:559–564; 1968. 20. Beutler, E. The glutathione instability of drug-sensitive red cells. A new method for the in vitro detection of drug sensitivity. J. Lab. Clin. Med. 49:84–95; 1957. 21. Matkovics, B.; Novak, Z.; Hoang, D. H.; Szabo, L.; Varga, S. J.; Zalesna, G. A comparative study on some important experimental animal peroxide metabolism enzymes. Comp. Biochem. Physiol. 56:31–34; 1987. 22. Misra, H. P.; Fridovich, I. The role of superoxide anion in the oxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247:3170–3175; 1972. 23. Sedlak, I.; Lindsay, R. H. Estimation of total, protein-bound and nonprotein sulphydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25:192–205; 1968. 24. Chin, D. T. Y.; Stults, F. H.; Tappal, A. L. Purification and properties of rat lung soluble glutathione peroxidase. Biochim. Biophys. Acta 445:558–566; 1976.
25. Placer, Z. A.; Cusman, L.; Johnson, B. C. Estimation of product of lipid peroxidation by malonyl dialdehyde in biochemical systems. Anal. Biochem. 16:359–364; 1966. 26. Tu´ri, S.; Ne´meth, I.; Varga, I.; Matkovics, B.; Dobos, E´. Erythrocyte defense mechanism against free oxygen radicals in haemodialysed uremic children. Pediatr. Nephrol. 5:179– 183; 1991. 27. Lowry, O. H.; Rosebrough, N. I.; Farr, A. L.; Randal, R. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 28. Szebeni, J.; Winterbourn, C. C.; Carrell, R. W. Oxidative interactions between haemoglobin and membrane lipid. A liposome model. Biochem. J. 220:685–692; 1984. 29. Andreoli, S. P. Mechanism of oxidant mediated cell injury. Pediatr. Nephrol. 9:C27; 1995. 30. McCord, J. M.; Roy, R. S. The pathophysiology of superoxide: Roles in inflammation and ischemia. Can. J. Physiol. Pharmacol. 60:1346–1352; 1982. 31. Ne´meth, I.; Boda, D. Blood glutathione redox ratio as a parameter of oxidative stress in premature infants with IRDS. Free Radic. Biol. Med. 16:347–353; 1994.
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PII S0891-5849(96)00284-5
Original Contribution OXIDATIVE STRESS AND ANTIOXIDANT DEFENSE MECHANISM IN GLOMERULAR DISEASES Sa´ndor Tu´ri,* Ilona Ne´meth,* Attila Torkos,* Levente Sa´ghy,* Ilona Varga,† Be´la Matkovics,† and Judit Nagy‡ *Department of Paediatrics, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; †Department of Biology, Attila Jo´zsef University, Szeged, Hungary; and ‡2nd Department of Medicine, Medical University, Pe´cs, Hungary (Received 21 November 1995; Revised 26 April 1996; Accepted 9 May 1996)
Abstract—The changes in red blood cell (RBC) lipid peroxidation [measured via the malonyl dialdehyde (MDA) concentration], reduced (GSH), and oxidized glutathione (GSSG) levels, hemoglobin (Hb) oxidation and antioxidant enzyme [catalase (Cat), glutathione peroxidase, and superoxide dismutase (SOD)] activities were studied in 45 pediatric patients with various glomerular diseases [minimal change nephrotic syndrome (MCNS) in relapse or in remission, lupus nephropathy (SLE), poststreptococcal glomerulonephritis (APSGN), IgA nephropathy (IGA gn)], and in 20 adult patients with IGA gn and also in 15 pediatric and 14 adult controls. The in vitro effects of hydrogen peroxide [acetyl phenylhydrazine (APH) test] on the GSH and Hb metabolisms were likewise investigated. There was an increased oxidative stress in MCNS with relapse, IGA gn, SLE gn, and APSGN, which could be detected in the GSH and Hb oxidation and in the lipid peroxidation on the peripheral RBC-s. The RBC SOD and Cat activities were significantly lower in all patients than in the controls. The RBC GSSG level was significantly elevated in all patients, with the exception of MCNS in remission. This stimulated a compensatory GSH production in MCNS with relapse and in IGA gn, but not in SLE or APSGN. The regeneration of GSH from GSSG was reduced in MCNS with relapse, SLE, and IGA gn, but not in APSGN. In remission, the GSH-GSSG redox system normalizes, but in vitro the APH test stimulates an intensive Hb oxidation. In conclusion, there is a correlation between the presence of active glomerular disease and the evidence of oxidative changes in the various parameters measured in peripheral RBCs. Copyright q 1996 Elsevier Science Inc. Keywords—Antioxidant defense, Glomerular disease, Oxidative stress, Free radicals
puromycin aminonucleoside (PAN)-induced nephrosis,5,6 murine lupus nephritis,7 and adriamycin-induced nephrosis.8 However, the role of oxidative injury in glomerular diseases was not confirmed by other groups.9,10 The study by Alfrey et al. indicated that the increased urine iron excretion is associated with hydroxyl radical production, which causes tubular injury.11 Vitamin E treatment was earlier found to cause an increased renal protein SH production, a decreased protein excretion, and an improved creatinine clearance in the heterologous phase of antiGbm nephritis.12 A selenium-deficient diet resulted in a reduced glutathione peroxidase activity (GP-ase) and an increased urine protein excretion.13 Fish oil feeding of rats with murine lupus nephritis resulted in an undetectable level of proinflammatory cytokines and an increased antioxidant enzyme gene ex-
INTRODUCTION
A still growing body of evidence has accumulated, indicating that a disturbance of the balance between oxidative stress and antioxidant defense mechanisms plays a major role in the pathomechanism of glomerular diseases. In antiglomerular basement membrane nephritis (anti-Gbm nephritis), the plasma lipid peroxidation is increased,1 and the administration of antioxidant enzymes [superoxide dismutase (SOD) and catalase (Cat)] resulted in a reduction of renal malonyl dialdehyde (MDA) production and urine protein excretion; further morphological protection was detected.2–4 Similar findings were observed in Address correspondence to: Sa´ndor Tu´ri M.D., Department of Paediatrics Albert Szent-Gyo¨rgyi Medical University, Kora´nyi Street 14, 6725 Szeged, Hungary. 161
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S. TURI et al. Table 1. MDA Concentrations in the Plasma and RBC (mean { SD)
PATIENTS AND METHODS
nM MDA/mg protein Dg
n
Cont.ped. Cont.adult APSGN SLE MCNS rel. MCNS rem. IGA ped. IGA adult
15 14 8 7 15 17 4 20
plasma 0.189 0.202 0.257 0.244 0.332 0.196 0.240 0.244
{ { { { { { { {
RBC
0.017 0.019 0.055* 0.044* 0.030† 0.053 0.046* 0.041*
0.350 0.356 0.486 0.379 0.454 0.439 0.430 0.438
{ { { { { { { {
0.030 0.025 0.010† 0.016* 0.010† 0.012† 0.060* 0.070*
APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * p õ 0.05. † p õ 0.01.
pression.14 The results of Andreoli et al.15 and Yagucchi et al.16 suggested that reactive oxygen species could be induced by glomerular resident cells. It appeared that infiltration of neutrophils in the glomeruli might not be related to proteinuria and glomerular injuries in the first phase of Masugi nephritis. It was postulated that massive proteinuria in the first phase of Masugi nephritis might be correlated with the activities of reactive oxygen species induced by the glomerular cells.16 Apart from these experimental results, only scanty clinical data are available on the roles of oxidative stress and antioxidant defense mechanisms in the pathogenesis of glomerular diseases. The aim of the present study was to investigate the changes in lipid peroxidation, the red blood cell (RBC) glutathione redox system, hemoglobin (Hb) oxidation and antioxidant enzyme activities in glomerular diseases of pediatric and adult patients.
Forty-five pediatric (25 boys and 20 girls) and 20 adult patients (12 males and 8 females) were investigated in comparison with 15 pediatric and 14 adult age and sex-matched controls. The median ages of the patients were as follows: children 8 years (range 3–15 years), adults 31 years (21–45 years). The distribution of the clinical subgroups among the pediatric patients: acute poststreptococcal glomerulonephritis (APSGN): 8; systemic lupus erythematodes associated with nephrotic syndrome (SLE): 7; minimal change nephrotic syndrome in relapse (MCNS rel): 15; minimal change nephrotic syndrome in a remission of õ2 months’ duration (MCNS rem): 17; IgA nephropathy (IGA ped.): 4 patients. All of the adult patients had IgA nephropathy (IGA adult). The nephrotic patients (MCNS rel., IGA ped. or SLE) displayed ú 2 g protein excretion daily. Individuals belonging in the APSGN and IGA adult subgroups had ú 200 RBC-s/ml urine. Seven adult IGA patients exhibited ú 2 g/d urine protein excretion. Ten members of the adult IGA subgroup required antihypertensive therapy (calcium channel blockers and/or converting enzyme inhibitors). The pediatric patients received no antihypertensive treatment. The nephrotic children (MCNS rel. and IGA) participated in Prednisolone therapy (60 mg/m2 body surface area daily in the first month, and 35 mg/m2 every second day in the second month). The SLE patients received chlorambucil (0.2 mg/kg body weight daily), or azathioprine (2 mg/kg/d) together with low-dose Prednisolone. None of the patients were given nonsteroidal antiinflammatory drugs, albumin or blood transfusions in the 2-week period prior to the investigation. The study was approved by the University Research Ethical Committee. After informed consent had been obtained, blood
Table 2. RBC Antioxidant Enzyme Activities (mean { SD) U/mg protein 1 1002 Dg
n
Cont.ped Cont.adult APSGN SLE MCNS rel. MCNS rem. IGA ped. IGA adult
15 14 8 7 15 17 4 20
GP-ase 0.91 1.02 1.06 0.81 0.89 0.91 0.90 0.89
{ { { { { { { {
0.06 0.06 0.3 0.07 0.06 0.21 0.10 0.14
SOD 1.32 1.30 0.256 0.220 0.553 0.213 0.337 0.348
{ { { { { { { {
0.12 0.32 0.018* 0.08* 0.39* 0.13* 0.03* 0.02*
Cat 9.48 10.2 5.14 5.67 6.60 5.52 5.64 5.61
{ { { { { { { {
1.9 1.4 1.9* 1.8* 0.9* 2.03* 1.1* 1.7*
APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * p õ 0.01.
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Fig. 1. Red blood cell oxidized glutathione (GSSG) concentrations in different glomerular diseases (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IgA nephropathy; ped.: pediatric. ** Å p õ 0.01.
samples were drawn into tubes with EDTA and heparin for the following studies.
with 5 mg/ml acetylphenylhydrazine (APH) at 377C for 1 h. The residual GSH was expressed as a percentage of the original concentration.
GSH determinations The concentrations of reduced (GSH) and oxidized glutathione (GSSG) in the whole blood hemolysate were measured by means of the highly sensitive method of Tietze.17 This assay responds to both GSH and GSSG, so GSSG was determined separately after the alkylation of GSH with N-ethylmaleimide (NEM) according to the method of Akerboom and Sies.18 GSSG was separated from NEM by gel filtration with Sephadex G-10 (Pharmacia, Uppsala, Sweden).19 Both GSH and GSSG were related to the Hb contents of the haemolysates.
Antioxidant enzyme and MDA studies Heparinized blood was used for MDA and antioxidant enzyme activity measurements in parallel with GSH and GSSG determinations. The methods for the measurement of SOD,21,22 Cat,23 GP-ase23,24 and MDA25 activities were described in detail previously.26 The protein concentration of RBC-s was determined with the Folin reagent.27 The appearance of Hb derivative
GSH instability test This was carried out by the method of Beutler.20 After GSH determination, whole blood was incubated
In in vitro tests, the changes in the level of hemichrome during oxidative stress were examined by the method of Szebeni et al.28 before and after APH incu-
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Fig. 2. Red blood cell reduced glutathione (GSH) levels in different glomerulopathies (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * Å p õ 0.05.
bation. All determinations were carried out in duplicate (two samples from each patient). Statistical analyses were performed with the paired t-test and Spearman rank correlation test. The results for the groups are given as mean values plus or minus SD.
A positive correlation was found between the RBC and plasma MDA concentrations (r Å 0.59, p õ 0.05) (Table 1). There were no significant differences in RBC GPase activities between patients and controls. However, the SOD and Cat values were significantly lower in all patients than in the controls (p õ 0.01) (Table 2).
RESULTS
MDA and antioxidant enzyme studies
RBC GSH-GSSG determinations
The RBC MDA results were elevated in all patient groups as compared with the controls (SLE, IGA ped., IGA adult p õ 0.05; MCNS rel., MCNS rem., APSGN p õ 0.01). Similarly, the plasma MDA values were also significantly higher in all patient groups (with the exception of MCNS rem) than in the controls (MCNS rel p õ 0.01, APSGN, SLE, IGA ped., IGA adult p õ 0.05).
As compared with the controls, the GSH oxidation (GSSG concentration) was significantly higher in all patient groups (p õ 0.01) with the exception of MCNS rem (Fig. 1). We observed a compensatory increase of GSH in the MCNS rel and IGA ped, groups as compared with the pediatric controls (p õ 0.05). Nevertheless, in APSGN this level was significantly lower (p õ 0.05).
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Fig. 3. Red blood cell glutathione redox ratio (GSSG/GSH %) in glomerular diseases (mean { SD). APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. ** Å p õ 0.01.
The other groups did not differ from their age-matched controls (Fig. 2). The GSSG/GSH ratio was significantly higher in APSGN, SLE, and MCNS rel. as compared with the other patient groups and the controls (p õ 0.01) (Fig. 3). In the APH test the GSH regeneration was significantly lower in the groups of SLE (p õ 0.001), MCNS rel. and IGA adult (p õ 0.05) than in the controls. In response to hydrogen hyperoxide, a significantly higher productions of hemichrome was observed in all patients’ groups (SLE p õ 0.001, APSGN, MCNS rel. p õ 0.01, MCNS rem., IGA adult and ped. p õ 0.05) than in the age-matched controls. It is noteworthy that the adult controls gave significantly higher values than the pediatric controls (p õ 0.05) (Fig. 4). DISCUSSION
Reactive oxygen molecules participate in the pathogenesis of various renal diseases, including inflamma-
tory lesions such as glomerulonephritis and interstitial nephritis, ischemic reperfusion injury, hemolytic uremic syndrome and toxic nephropathies, and possibly in the progression of chronic renal failure. These reactive oxygen species, including superoxide anion, hydrogen peroxide, hydroxyl radical, hypochlorous acid, and peroxynitrite, may be generated by activated neutrophils, monocytes, and mesangial cells during metabolic processes.29 Oxidant stress may induce injury to proteins by oxidation of critical amino acids, resulting in a loss of enzymatic activity or structural integrity. Oxidant-induced lipid peroxidation causes a loss of membrane stability and integrity, leading to an increased transepithelial permeability. The mechanisms of cell injury that lead to cell death following oxidant stress in cells are complex and multifactorial and may differ according to cell type. We have demonstrated an increased oxidative stress in IGA gn, SLE, APSGN, and in MCNS with relapse, which was detected in the GSH and Hb oxidation on peripheral RBC-s and lipid
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Fig. 4. The changes of reduced glutathione stability and hemoglobin oxidation after an in vitro oxidation stress. APSGN: acute poststreptococcal glomerulonephritis; SLE: lupus nephropathy; MCNS: minimal change nephrotic syndrome; rem.: remission; rel.: relapse; IGA: IGA nephropathy; ped.: pediatric. * Å p õ 0.05, ** Å p õ 0.01, *** Å p õ 0.001, § Å p õ 0.05 in controls.
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peroxidation in RBC-s and plasma. There was no difference in GSH and Hb oxidation and lipid peroxidation among these various forms of active glomerular diseases. The superoxide radical plays a major role in the neutrophil-mediated acute inflammatory response. Neutrophils produce superoxide for the primary purpose of aiding in the killing of microbes. The superoxide released from actively phagocytosing neutrophils serves to attract more neutrophils by reacting with and activating a latent chemotactic factor present in the plasma30 and results in extensive tissue damage. By preventing the activation of this superoxide-dependent chemotactic factor, SOD exerts potent antiinflammatory action. It is very likely that our MCNS patients produce the superoxide radical via their glomerular cells. In our study, the RBC SOD and Cat activities were significantly lower in all patients, including MCNS in rem, than in the controls. The increased GSH oxidation stimulates a compensatory GSH production in MCNS rel and IGA gn, but not in SLE or APSGN. The GSSG/ GSH ratio was the highest in SLE, APSGN and MCNS rel, which reflects an increased oxidative stress. The blood glutathione redox ratio is a parameter of the oxidative stress.31 The regeneration of GSH from GSSG was reduced in MCNS rel, SLE, and adult IGA gn. Simultaneously, an increase in the hemichrome percentage was observed in the same groups of patients. The insufficient regeneration of GSH resulted in an increased production of irreversibly oxidized Hb. The MCNS patients in remission featured a markedly decreased GSSG production and GSSG/GSH redox ratio. In the APH test the GSH stability improved and the percentage of hemichrome decreased resulting in a better Hb protection. This relates to a more specific marker role of the GSH system, in measuring the activity of oxidative stress. As far as the role of Prednisolone, or the other immunosuppressive drugs (chlorambucil and azathioprine) in the increased oxidative stress is concerned, further studies are required. At least no correlation was observed between the dose of these drugs and the level of GSH and Hb oxidation or MDA concentration. APSGN patients had similar results as the nephrotic patients treated with immunosuppressive therapy. Lastly, MCNS patients in remission and without Prednisolone had similarly elevated level of RBC MDA and low activity of RBC SOD and Cat as it was observed in MCNS with relapse. These clinical data yield multiple evidence that oxidative stress is present in the development of glomerular disease, although there are differences between the histological groups in the compensatory reactions of
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the GSH-GSSG redox system. In MCNS the source of the free oxygen radicals could be glomerular cells, while in the other forms the role of activated neutrophils or mononuclear cells cannot be excluded. In conclusion, antioxidant therapy and diet might be beneficial in the management of these glomerular diseases. Acknowledgements — This study was supported by Hungarian ETT (525/9306) and OTKA (T01 7476) research grants.
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